Methods for treating gastrointestinal diseases

ABSTRACT

Described herein are macrolide and ketolide antibiotics and pharmaceutical compositions, methods, and uses thereof for treating gastrointestinal diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuing application under 35 U.S.C. §120 ofU.S. patent application Ser. No. 13/125,551 filed Apr. 21, 2011, nowU.S. Pat. No. 8,791,080 issued on Jul. 29, 2014, which is a U.S.national entry application under 37 C.F.R. §371(b) of PCT InternationalApplication Ser. No. PCT/US2009/061976 filed Oct. 24, 2009, which claimspriority under 35 USC §119(e) to U.S. Provisional Application Ser. No.61/108,110, filed on Oct. 24, 2008, U.S. Provisional Application Ser.No. 61/108,112, filed on Oct. 24, 2008, U.S. Provisional ApplicationSer. No. 61/108,134, filed on Oct. 24, 2008, U.S. ProvisionalApplication Ser. No. 61/108,137, filed on Oct. 24, 2008, U.S.Provisional Application Ser. No. 61/108,168, filed on Oct. 24, 2008, andU.S. Provisional Application Ser. No. 61/162,109, filed on Mar. 20,2009, the entire disclosure of each of which are incorporated herein byreference.

TECHNICAL FIELD

The invention described herein relates to the treatment ofgastrointestinal diseases. In particular, the invention described hereinrelates to the treatment of gastrointestinal diseases with macrolide andketolide antibiotics.

BACKGROUND AND SUMMARY OF THE INVENTION

Most of the bacteria in the small intestine are Gram-positive, whilethose in the colon are mostly Gram-negative. The first part of the colonis mostly responsible for fermenting carbohydrates, while the latterpart mostly breaks down proteins and amino acids. Bacterial growth israpid in the cecum and ascending colon, which has a low pH, andcorrespondingly slow in the descending colon, which has an almostneutral pH. The body maintains the proper balance and locations ofspecies by altering pH, the activity of the immune system, andperistalsis.

Gastritis is an inflammation of the lining of the stomach. There aremany possible causes. Gastritis may be caused by excessive alcoholconsumption, or the prolonged use of nonsteroidal anti-inflammatorydrugs, also known as NSAIDs, such as aspirin or ibuprofen. Sometimesgastritis develops after major surgery, traumatic injury, burns, orsevere infections. Certain diseases, such as pernicious anemia andchronic bile reflux, or autoimmune disorders, can cause gastritis aswell. Importantly, gastritis may be caused by infection with bacteria,such as Helicobacter pylori. The most common symptom is abdominal upsetor pain. Other symptoms are indigestion, abdominal bloating, nausea, andvomiting, or a feeling of fullness or burning in the upper abdomen.

Gastroenteritis is inflammation of the gastrointestinal tract, involvingboth the stomach and the small intestine, often resulting in acutediarrhea. The inflammation is caused most often by infection withcertain viruses, but may also be caused by bacteria or parasites.Worldwide, inadequate treatment of gastroenteritis kills 5 to 8 millionpeople per year, and is a leading cause of death among infants andchildren under 5. Similarly, enteritis refers to inflammation of thesmall intestine with similar causes.

Many different bacteria can cause gastroenteritis, including Salmonella,Shigella, Staphylococcus, Campylobacter jejuni, Clostridium, Escherichiacoli, Yersinia, and others. Some sources of the infection are improperlyprepared food, reheated meat dishes, seafood, dairy, and bakeryproducts. Each organism causes slightly different symptoms but allresult in diarrhea. Colitis, inflammation of the large intestine, mayalso be present.

Several Salmonella species are capable of causing gastroenteritis,including S. enterica, which is subdivided into several serovars.Illustrative examples include Serovar Typhi (previously known as S.Typhi), which is the disease agent responsible for typhoid fever, andSerovar Typhimurium (also known as S. Typhimurium), which leads to aform of human gastroenteritis sometimes referred to as salmonellosis.Several Shigella species are also responsible for gastroenteritis.Illustrative examples include S. boydii; S. dysenteriae, which is amajor cause of dysentery; S. flexneri; and S. sonnei. An illustrativespecies of Campylobacter causing gastroenteritis in humans is C. jejuni.It is one of the most common causes of human gastroenteritis in theworld. Yersinia species are also a cause of gastroenteritis, anillustrative example is the zoonotic Y. enterocolitica. The diseasecaused by Y. enterocolitica is called yersiniosis. Some strains ofHelicobacter are pathogenic to humans and are strongly associated withpeptic ulcers, chronic gastritis, duodenitis, and stomach cancer. Anillustrative species responsible for disease in humans is H. pylori.

Reportedly clarithromycin (CLR) is the only known macrolide antibioticthat works in vivo on Helicobacter pylori. Although other macrolideantibiotics show in vitro activity against H. pylori, there often arenot sufficiently active at low pH to work in vivo. In addition, mayantibiotics, such as azithromycin (AZI) and telithromycin (TEL), do notachieve sufficiently high tissue and blood circulating levels to showefficacy on H. pylori. Without being bound by theory, it is suggestedherein that high protein binding may prevent other macrolide antibioticsfrom having such in vivo activity. Ordinarily, a minimum pH is requiredfor macrolide antibiotics to show activity.

It has been surprisingly discovered herein that triazole-containingmacrolides, including ketolides, exhibit high anti-bacterial activity atlow pH, or at pH levels much lower than the minimum pH required by othermacrolide antibiotics for efficacy. It has also been unexpectedlydiscovered herein that the compounds described herein have highanti-bacterial activity against gastroenteritis disease pathogens (GDP),such as H. pylori (HP), and gastritis and diarrheal illness pathogens,such as Camplylobacter jejuni (CJ), Salmonella spp. (SAL) and Shigellaspp. (SHI).

In one illustrative embodiment, compounds of Formula (I) are describedherein

including pharmaceutically acceptable salts, hydrates, solvates, esters,and prodrugs thereof.

In one aspect, R₁₀ is hydrogen or acyl. In another aspect, X is H; and Yis OR₇; where R₇ is a monosaccharide or disaccharide, alkyl, aryl,heteroaryl, acyl, or C(O)NR₈R₉, where R₈ and R₉ are each independentlyselected from the group consisting of hydrogen, hydroxy, alkyl,arylalkyl, alkylaryl, heteroalkyl, aryl, heteroaryl, alkoxy,dimethylaminoalkyl, acyl, sulfonyl, ureido, and carbamoyl; or X and Yare taken together with the attached carbon to form carbonyl.

In another aspect, V is C(O), C(═NR₁₁), CH(NR₁₂, R₁₃), or N(R₁₄)CH₂,where N(R₁₄) is attached to the C-10 carbon of the compounds of Formulae1 and 2; wherein R₁₁ is hydroxy or alkoxy, R₁₂ and R₁₃ are eachindependently selected from the group consisting of hydrogen, hydroxy,akyl, arylalkyl, alkylaryl, alkoxy, heteroalkyl, aryl, heteroaryl,dimethylaminoalkyl, acyl, sulfonyl, ureido, and carbamoyl; R₁₄ ishydrogen, hydroxy, alkyl, aralkyl, alkylaryl, alkoxy, heteroalkyl, aryl,heteroaryl, dimethylaminoalkyl, acyl, sulfonyl, ureido, or carbamoyl.

In another aspect, W is H, F, Cl, Br, I, or OH.

In another aspect, A is CH₂, C(O), C(O)O, C(O)NH, S(O)₂, S(O)₂NH,C(O)NHS(O)₂. In another aspect, B is (CH₂)_(n) where n is an integerranging from 0-10, or B is an unsaturated carbon chain of 2-10 carbons.In another aspect, C is hydrogen, hydroxy, alkyl, aralkyl, alkylaryl,alkoxy, heteroalkyl, aryl, heteroaryl, aminoaryl, alkylaminoaryl, acyl,acyloxy, sulfonyl, ureido, or carbamoyl.

In another embodiment, compositions including a therapeuticallyeffective amount of one or more compounds of formula (I), or the varioussubgenera thereof are described herein. The pharmaceutical compositionsmay include additional pharmaceutically acceptable carriers, diluents,and/or excipients.

In another embodiment, methods are described herein for treatingdiseases arising from pathogenic organism populations causing enteritis,gastroenteritis, and/or a related disease. The methods include the stepof administering a therapeutically effective amount of one or morecompounds of formula (I), or the various subgenera thereof are describedherein, to a patient in need of relief or suffering from a diseasecaused by a pathogenic organism.

In another embodiment, uses are described herein for the manufacture ofmedicaments. The medicaments include a therapeutically effective amountof one or more compounds of formula (I), or the various subgenerathereof are described herein, or one or more compositions thereofdescribed herein. The medicaments are suitable for treating diseases,such as enteritis, gastroenteritis, and/or a related disease arisingfrom pathogenic organism populations.

In another embodiment, compounds, compositions, methods, and medicamentsare described herein for treating diseases caused by H. pylori.

Each embodiment of the compositions, methods, and medicaments include atherapeutically effective amount of one or more triazole-containingmacrolides or ketolides, such as one or more compounds or formula (I).The therapeutically effective amount is administered to a patient inneed of relief or suffering from the disease.

In another embodiment, compounds, compositions, methods, and medicamentsare described herein for treating diseases caused by H. pylori. thatinclude the co-administration of one or more proton pump inhibitors,such as but not limited to omeprazole, esopremazole, and the like.

In another embodiment, oral formulations of the compounds andcompositions described herein include enteric coating. Without beingbound by theory, it is believed herein that enteric coating may preventstomach acid degradation of proton pump inhibitors to achiralintermediates.

In another embodiment, compounds, compositions, methods, and medicamentsare described herein for treating enteritis, gastroenteritis, andrelated diseases, that include the co-administration of otherantibiotics, including but not limited to fluoroquinolone antibiotics,metronidazoles, vancomycin, and the like, and combinations thereof.

In another embodiment, compounds, compositions, methods, and medicamentsare described herein for treating enteritis, gastroenteritis, andrelated diseases, accompanied by diarrhea symptoms, that include theco-administration of other compounds for decreasing gut motility. It isappreciated that the macrolide compounds described herein may alsodecrease gut motility. Illustrative gut motility decreasing agentsinclude but are not limited to loperamide, an opioid analogue commonlyused for symptomatic treatment of diarrhea, and bismuth subsalicylate(BSS), an insoluble complex of trivalent bismuth and salicylate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparative susceptibilities of S. aureus ATCC 25923 and L.monocytogenes EGD to CEM-101, TEL, AZI, and CLR, based on MICdeterminations in pH-adjusted broth.

FIG. 2. Short-term time-kill effect of CEM-101 and AZI on S. aureus(ATCC 25923) in broth (left panels; pH 7.4) or after phagocytosis byTHP-1 macrophages (right panels). Both drugs were used at anextracellular concentration of either 0.7 (top panels) or 4 (bottompanels) mg/liter. MICs of CEM-101 and AZI were 0.06 and 0.5 mg/liter,respectively. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 3. Concentration-effect relationships for CEM-101, TEL, CLR, andAZI toward S. aureus (ATCC 25923) in broth (left panels) and afterphagocytosis by THP-1 macrophages (right panels). The ordinate shows thechange in CFU (Δ log CFU) per ml (broth) or per mg of cell protein(THP-1 macrophages) at 24 h compared to the initial inoculum. Theabscissa shows the concentrations of the antibiotics as follows: (i) toppanels, weight concentrations (in mg/liter) in broth (left) or in theculture medium (right) and (ii) bottom panels, multiples of the MIC asdetermined in broth at pH 7.4. All values are means±standard deviations(SD) of three independent experiments (when not visible, SD bars aresmaller than the symbols). Statistical analysis based on global analysisof curve-fitting parameters (one-way analysis of variance); the onlysignificant difference is between CEM-101 and AZI in broth (P=0.04).Numerical values of the pertinent pharmacological descriptors andstatistical analysis of their differences are shown in Table 1.

FIG. 4. Concentration-effect relationships for CEM-101 and AZI towardintraphagocytic L. monocytogenes (strain EGD, left panels) and L.pneumophila (strain ATCC 33153, right panels). The ordinate shows thechange in CFU (Δ log CFU) per mg of cell protein at 24 h (L.monocytogenes) or 48 h (L. pneumophila) compared to the initialpostphagocytosis inoculum. The abscissa shows the concentrations of theantibiotics as follows: (i) top panels, weight concentrations (inmg/liter); (ii) bottom panels, multiples of the MIC as determined inbroth at pH 7.4. All values are means±standard deviations (SD) of threeindependent experiments (when not visible, SD bars are smaller than thesymbols).

FIG. 5. Accumulation of CEM-101 versus comparators in THP-1 cells at 37°C. (all drugs at an extracellular concentration of 10 mg/liter). (A)Kinetics of accumulation (AZI); Cc, intracellular concentration; Ce,extracellular concentration); (B) influence of the pH of the culturemedium on the accumulation (30 min) of CEM-101 (solid symbols and solidline) and AZI (open symbols and dotted line); (C) influence of monensin(50 μM; 2-h incubation), verapamil (150 μM; 24-h incubation), orgemfibrozil (250 μM; 24-h incubation) on the cellular accumulation ofAZI and CEM-101. All values are means±standard deviations (SD) of threeindependent determinations (when not visible, SD bars are smaller thanthe symbols).

FIG. 6. Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages. Bars represent the MICs (in mg/L) or theextracellular static dose.

FIG. 7. Intracellular Activity of CEM-101 compared to AZI, CLR, and TEL,expressed as a dose response curve of Δ log CFU from time 0 to 24 hoursversus log dose.

DETAILED DESCRIPTION

In one embodiment, compositions, methods, and medicaments are describedherein for treating diseases that are caused at least in part by GDPstrains, where the compositions, methods, and medicaments include atherapeutically effective amount of a triazole-containing macrolide orketolide compound described herein. In another embodiment, compositions,methods, and medicaments are described herein for treating CLR-resistant(CLR-R) gastric diseases, where the compositions, methods, andmedicaments include a therapeutically effective amount of atriazole-containing macrolide or ketolide compound described herein.

In another embodiment, compounds are described herein that are activeintracellularly. It has also been discovered herein that theintracellular accumulation and intracellular activity oftriazole-containing macrolides was not affected by Pgp or MultidrugResistant Protein (MRP) inhibitors. Accordingly, it is believed that thecompounds described herein are not substrates or are poor substrates ofP-glycoprotein (plasma or permeability gycoprotein, Pgp). It isappreciated that Pgp is an efflux mechanism that may lead to resistanceby some organisms against certain antibiotics, such as has been reportedfor AZI and ERY in macrophages in which both antibiotics are substratesof the P-glycoprotein. Accordingly, it has been surprisingly found thatthe compounds described herein accumulate intracellulary. In addition tothe intracellular accumulation, it has been surprisingly discovered thatthe triazole-containing macrolide and ketolide compounds describedherein have high intracellular activity. It has also been surprisingfound herein that the compounds described herein have lower proteinbinding than is typical for macrolides at lower pH, such as the pH foundin bacterial infections, including but not limited to abscesses. It isappreciated that the lack of intracellular activity typically observedwith anti-bacterial agents, including other macrolides and ketolides,may be due to high protein binding, and/or to the relatively lower pH ofthe intracellular compartments, such as is present in abscesses.

However, even when not removed by active efflux, the concentration ofother anti-bacterial agents, including other macrolides and ketolides,in macrophages may not be efficacious in treating disease because of thelow pH of the lysozomal compartment. For example, the acidic environmentprevailing in the phagolysosomes (where S. aureus sojourns during itsintracellular stage) may impair the activity of antibiotics, such as theAZI, CLR and TEL. It has been unexpectedly found that the compoundsdescribed herein retain their anti-bacterial activity at low pH. It isappreciated that the intracellular activity of the compounds describedherein may be an important determinant for fast and complete eradicationand, probably also, for prevention of resistance in the target organism.

Lack of effective antimicrobial therapy results in intracellularsurvival of bacteria, which remains a major cause of bacterialspreading, life-threatening therapeutic failures, and establishment ofchronic, relapsing infections. These situations are observed during thecourse of infections caused by many organisms causing gastrointestinaldiseases, including H. pylori, C. jejuni, Salmonella, and Shigella.

While it has been reported that intracellular accumulation of anantibiotic is indicative of efficient activity against bacteria,pharmacodynamic evaluation of a large series of commonly usedantibiotics has revealed that other parameters such as intracellularbioavailability and modulation of activity in the infected compartmentare also important. The observations described herein confirm and extendprevious observations made with macrolides in this context due to thesurprising differential behavior exhibited by the triazole-containingmacrolides described herein, compared to known macrolide and ketolides,such as TEL, AZI, and CLR.

It is surprisingly found that triazole-containing macrolides accumulateto a considerably larger extent than the comparators, including AZI, andconsistently expresses greater potency (decreased values of E₅₀ andC_(s)) while showing similar maximal efficacy (E_(max)) to comparators.Without being bound by theory, it is believed that this indicates thatthe improvements resulting from the structural modifications introducedin CEM-101 relate to modulation of pharmacokinetic properties andintrinsic activity (including its reduced susceptibility tophysico-chemical conditions prevailing in the infected compartment)rather than to a change in its mode of action. Thus, triazole-containingmacrolides exhibit the essentially bacteriostatic character ofmacrolides, but express it better in the intracellular milieu and atconsiderably lower extracellular concentrations than the comparators.

Without being bound by theory, it is believed that the cellularaccumulation of triazole-containing macrolides, such as CEM-101, resultsfrom the general mechanism of proton trapping of weak organic basesenvisaged for all macrolides as accumulation is almost completelysuppressed, in parallel with AZI, by exposure to acid pH or to theproton ionophore monensin. Based on the general model ofdiffusion/segregation of weak bases in acidic membrane-boundcompartments, accumulation is determined by the number of ionizablegroups and the ratios between the membrane permeability coefficients ofthe unionized and ionized forms of the drug. While CEM-101 has twoionizable functions, the pKa of the aminophenyltriazole is calculated tobe less than 4, suggesting that the molecule is largely monocationic(similar to CLR and TEL) at neutral and even at lysosomal pH (˜5). Incontrast, AZI has two ionizable functions with pK_(a)s>6 and istherefore dicationic intracellularly. CEM-101, however, possesses afluoro substituent in position 2, which should make it more lipophilicthan CLR or TEL. Without being bound by theory, it is believed that theratio of the permeability constants of the unionized and ionized formsof CEM-101 in comparison with LR or TEL may be as important as thenumber of ionizable functions to determine the level of cellularaccumulation of weak organic bases. Without being bound by theory, it isbelieved that the greater cellular accumulation of CEM-101 may bepartially due to its lack of susceptibility to Pgp-mediated efflux(which is expressed by THP-1 macrophages under our culture conditions)in contrast to AZI.

It has been observed that many known macrolides have a large volume ofdistribution, which it is believed is related to their ability toaccumulate inside eukaryotic cells by diffusion/segregation in acidiccompartments, namely lysosomes and related vacuoles. As a consequence,known macrolides had been considered candidates for the treatment ofinfections localized in these compartments. Thus, it might be assumedthat macrolides are suitable for treating infections caused by typicalintracellular pathogens. However, direct quantitative comparisonsbetween intracellular and extracellular activities using facultativeintracellular pathogens, such as S. aureus or L. monocytogenes, suggestthat known macrolides express only a minimal fraction of theirantibacterial potential intracellularly, especially considering theirgreat intracellular accumulation. This minimized antibacterial potentialagainst organisms replicating in phagolysosomes and related vacuoles isbelieved to be related to acidic pH which is known to reduce theactivity of known macrolides. Another factor is that some organisms,such as H. pylori, C. jejuni, Salmonella, and Shigella, may actuallyreplicate in other subcellular compartments. In addition, certainmacrolides, such as AZI, are subject to active efflux from macrophages,which further contributes to suboptimal intracellular activity.

In contrast, the cellular accumulation and intracellular activity of thetriazole-containing compounds described herein, using models that havebeen developed for the study of the intracellular pharmacodynamics ofantibiotics, is substantially improved over known macrolides, includingketolides. Thus, the compounds described herein maintain the maximalefficacy of their MICs, and show greater potency against intracellularforms of for example, against gastrointestinal disease causingorganisms, including H. pylori, C. jejuni, Salmonella, and Shigellacompared to TEL, AZI, and CLR. Without being bound by theory, it isbelieved that this improved intracellular potency of thetriazole-containing compounds described herein results from thecombination of the higher intrinsic activity against gastrointestinaldisease causing organisms, including H. pylori, C. jejuni, Salmonella,and Shigella coupled with the retained activity at low pH, and theability to distribute to a wide variety of intracellular compartments.

In another embodiment, the triazole-containing macrolide and ketolidecompounds have intracellular activity, such as intracellular activityagainst gastrointestinal disease causing organisms, including H. pylori,C. jejuni, Salmonella, and Shigella. It is appreciated that routinesusceptibility testing are usually determined against extracellularbacterial only, and therefore may be misleading in their prediction ofefficacy against intracellular organisms. In another embodiment,compounds, compositions, methods, and uses are described herein fortreating a disease caused at least in part by an intracellular H.pylori, C. jejuni, Salmonella, and/or Shigella. In another embodiment,the disease caused by the Staphylococcus infection is a gastrointestinaldisease. It is further appreciated that H. pylori, C. jejuni,Salmonella, and/or Shigella may include virulent strains, and thustreatment with bacteriostatic agents may be ineffective. For example,recurrence may be a problem when treating such strains. It has beenunexpectedly discovered herein that the compounds described herein arealso bactericidal and therefore useful in treating diseases caused bysuch strains of pylori, C. jejuni, Salmonella, and/or Shigella.

In another embodiment, the compounds, methods, and medicaments describedherein include a therapeutically effective amount of one or morecompounds described herein, wherein the therapeutically effective amountis an amount effective to exhibit intracellular antibacterial activity.

In another embodiment, compounds are described herein that arebactericidal. In another embodiment, the compounds, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidalactivity, including in vivo bactericidal activity. It has been reportedthat macrolides are generally bacteriostatic. Bacteriostatic compoundsdo not kill the bacteria, but instead for example inhibit growth andreproduction of bacteria without killing them; killing is accomplishedby bactericidal agents. It is understood that bacteriostatic agents mustwork with the immune system to remove the microorganisms from the body.Bacteriostatic antibiotics may limit the growth of bacteria via a numberof mechanisms, such as by interfering with bacterial protein production,DNA replication, or other aspects of bacterial cellular metabolism. Incontrast, bactericidal antibiotics kill bacteria; bacteriostaticantibiotics only slow their growth or reproduction. Penicillin is abactericide, as are cephalosporins, all belonging to the group ofβ-lactam antibiotics. They act in a bactericidal manner by disruptingcell wall precursor leading to lysis. In addition, aminoglycosidicantibiotics are usually considered bactericidal, although they may bebacteriostatic with some organisms. They act by binding irreversibly to30 s ribosomal subunit, reducing translation fidelity leading toinaccurate protein synthesis. In addition, they inhibit proteinsynthesis due to premature separation of the complex between mRNA andribosomal proteins. The final result is bacterial cell death. Otherbactericidal antibiotics include the fluoroquinolones, nitrofurans,vancomycin, monobactams, co-trimoxazole, and metronidazole.

In another embodiment, the compounds, compositions, methods, andmedicaments described herein include a therapeutically effective amountof one or more compounds described herein, wherein the therapeuticallyeffective amount is an amount effective to exhibit bactericidal activityagainst one or more gastrointestinal disease causing organisms,including H. pylori, C. jejuni, Salmonella, and Shigella. Without beingbound by theory, it is believed herein that treating such diseases usingbacteriostatic agents may be unsuccessful in two respects. First, simplystopping the progression of the disease with a bacteriostatic agent maybe insufficient because the immune system may not intervene to assist incuring the disease at a necessary level. For example, some bacterialorganisms are not killed by the immune system because they reside inintracellular compartments. Thus, once the treatment course has ended,rapid recurrence of disease may result. Second, because some portion ofthe bacterial population will likely be eliminated, the remainingpopulation may be selected for resistance development. It is believedherein that an intracellularly active agent, and/or an intracellularlyactive and bactericidal agent, will be efficacious in treating suchdiseases. In one illustrative embodiment, compounds described hereinthat achieve an intracellular concentration of 20× the MIC of thetargeted bacteria. It has been reported that most, if not all, macrolideantibiotics, though bactericidal in vitro, are only bacteriostatic invivo. For example, as described hereinbelow, when the time between thelast dose of compound was extended, the bioload reduction levelsremained the same for the triazole-containing compounds describedherein, indicating a bactericidal response. In contrast, the TEL and CLRdose groups demonstrated bioload increases when the time interval wasextended. Thus, those latter two macrolide/ketolide agents demonstrateda more classical bacteriostatic response.

In another illustrative embodiment, compounds of Formula (I) aredescribed herein where X and Y are taken together with the attachedcarbon to form a C(O) group. In another embodiment, X is H, Y is OR⁷,where R⁷ is a monosaccharide radical, such as cladinosyl. In anotherembodiment, compounds of Formula (I) are described herein where W isfluoro. In another embodiment, compounds of Formula (I) are describedherein where A and B are taken together to form an alkylene group,including but not limited to propylene, butylene, and pentylene. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form butylene. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene. In another embodiment, compounds of Formula(I) are described herein where A and B are taken together to formbutylenes and C is 2-pyridinyl or aminophenyl, such as 3-aminophenyl. Inanother embodiment, compounds of Formula (I) are described herein whereA and B are taken together to form propylenes, butylenes, or pentylenes;and C is aminophenyl, such as 3-aminophenyl. In another embodiment,compounds of Formula (I) are described herein where A and B are takentogether to form pentylene and C is 3-pyridinyl or benzotriazole. Inanother embodiment, compounds of Formula (I) are described herein whereC is an optionally substituted aryl or heteroaryl group. In anotherembodiment, compounds of Formula (I) are described herein where V is acarbonyl group. In another embodiment, compounds of Formula (I) aredescribed herein where R¹⁰ is hydrogen. In another embodiment, X is H, Yis OR⁷, where R⁷ is a monosaccharide radical, such as cladinosyl, and Cis 3-pyridinyl or benzotriazolyl.

In another embodiment, C is optionally substituted phenyl, such asphenyl, halophenyl, haloalkylphenyl, aminophenyl, and the like,optionally substituted pyridinyl, such as 2-pyridinyl and 3-pyridinyl,optionally substituted benzotriazole, and the like.

In another embodiment, A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group.

In another embodiment, compounds described in any of the precedingembodiments wherein V is C(O) are described. In another embodiment,compounds described in any of the preceding embodiments wherein W is Hor F are described. In another embodiment, compounds described in any ofthe preceding embodiments wherein A is CH₂, B is (CH₂)_(n), and n is aninteger from 2-4 are described. In another embodiment, compoundsdescribed in any of the preceding embodiments wherein C is aryl orheteroaryl are described. In another embodiment, compounds described inany of the preceding embodiments wherein C is 3-aminophenyl or3-pyridinyl are described. In another embodiment, compounds described inany of the preceding embodiments wherein R₁₀ is hydrogen. In anotherembodiment, compounds described in any of the preceding embodimentswherein A and B are taken together to form butylene or pentylene, and Xand Y are taken together with the attached carbon to form a C(O) group.In another embodiment, compounds described in any of the precedingembodiments wherein A and B are taken together to form butylene orpentylene, and X and Y are taken together with the attached carbon toform a C(O) group, and W is F.

In another embodiment, an antibacterial composition is described herein,wherein the composition includes an effective amount of one or morecompounds described herein, and a pharmaceutically acceptable carrier,excipient, or diluent therefor, or a combination thereof.

As used herein, the term “composition” generally refers to any productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationsof the specified ingredients in the specified amounts. Illustratively,compositions may include one or more carriers, diluents, and/orexcipients. The compounds described herein may be formulated in atherapeutically effective amount in conventional dosage forms for themethods described herein, including one or more carriers, diluents,and/or excipients therefor. Such formulation compositions may beadministered by a wide variety of conventional routes for the methodsdescribed herein in a wide variety of dosage formats, utilizingart-recognized products. See generally, Remington's PharmaceuticalSciences, (16th ed. 1980). It is to be understood that the compositionsdescribed herein may be prepared from isolated compounds describedherein or from salts, solutions, hydrates, solvates, and other forms ofthe compounds described herein. It is also to be understood that thecompositions may be prepared from various amorphous, non-amorphous,partially crystalline, crystalline, and/or other morphological forms ofthe compounds described herein.

The term “therapeutically effective amount” as used herein, refers tothat amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of the symptoms of thedisease or disorder being treated. In one aspect, the therapeuticallyeffective amount is that which may treat or alleviate the disease orsymptoms of the disease at a reasonable benefit/risk ratio applicable toany medical treatment. However, it is to be understood that the totaldaily usage of the compounds and compositions described herein may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically-effective dose level for anyparticular patient will depend upon a variety of factors, including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, gender and diet of the patient: the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidentally with the specific compound employed; andlike factors well known in the medical arts.

In one embodiment, the compounds described herein are administered to ahuman orally at a dose of about 1 to about 10 mg/kg, about 2 to about 8mg/kg, or about 4 to about 6 mg/kg of patient body weight. In anotherembodiment, the daily adult human dose is about 100 to about 1,000 mg,which may be administered qd, bid, tid, and the like. In anotherembodiment, the daily adult human dose is about 400 to about 600 mg,which may be administered qd, bid, tid, and the like. Such doses may beadministered, once, twice, or thrice per day. Illustrative oral unitdosages are 50, 100, 200, and 400 mg (single or divided). Without beingbound by theory, it is believed that such illustrative dosages aresufficient to achieve plasma levels of about 1 μg/mL, which may besufficient to observe bactericidal activity of the compounds describedherein, such as for H. pylori, C. jejuni, Salmonella, and Shigella. Itis appreciated that as described herein, the compounds described herein,including CEM-101, reach high concentration in tissues, such as lungtissues. Without being bound by theory, it is believed herein that thecompounds described herein, including CEM-101, may achieve tissue levelsthat are at least about 10-times the MIC for strains, includingmacrolide-resistant strains, such as but not limited to H. pylori, C.jejuni, Salmonella, and Shigella, including organisms that are resistantto macrolides or ketolides, such as AZI, TEL, and/or CLR.

The compounds described herein may be prepared as described herein, oraccording to US Patent Application Publication No. 2006/0100164 and inPCT International Publication No. WO 2009/055557, the disclosures ofwhich are incorporated herein by reference in their entirety.

Briefly, the synthesis of triazole containing ketolides begins with theknown two step preparation of the 12-acyl-imidazole intermediate 4(Scheme I) from clarithromycin (2). Intermediate 4 is converted into the11,12-cyclic carbamates 5a-c by the reaction with the corresponding 3-,4- or 5-carbon linked amino alcohols. Treatment of 5a-c with tosylchloride provides tosylates 6a-c. Displacement of the tosyl group withNaN₃ gives the corresponding azido compounds 7a-c. Cleavage of thecladinose sugar of 7a-c to 8a-8c is accomplished by treatment with HClin MeOH. Swern oxidation of the 3-hydroxy group of 8a-c gives thecorresponding protected ketolides 9a-c which are subsequentlydeprotected with methanol to afford the required azido ketolides 10a-c,respectively. These azido compounds were reacted withterminally-substituted alkynes in the presence of copper iodide intoluene at 60° C. to regio-selectively afford the corresponding4-substituted-[1,2,3]-triazoles 11a-18a, 11b-18b, and 11c-18c.

The azide of intermediates 10a-c is converted to the4-substituted-[1,2,3]-triazoles via a cycloaddition reaction withsubstituted acetylenes. Triazole rings may be formed via a Huisgen 1+3cycloaddition reaction between an azide and an alkyne resulting in amixture of 1,4- and 1,5-regioisomers as depicted in Route A of SchemeII. Alternatively, the procedure of Rostovtsev et al.⁸ may be followedusing the addition of a CuI catalyst to the reaction to selectively orexclusively produce the 1,4-regioisomer as depicted in Route B of SchemeII.

The triazole ring side chain is also incorporated into theclarithromycin ring system. In one embodiment, a butyl alkyl side chainis chosen. It is appreciated that many butyl side chain analogs in theketolide series have improved antibacterial activity based on in vitroMIC results. Intermediate 7b is directly converted into the4-substituted-[1,2,3]-triazole via copper catalyzed cyclization withterminally substituted acetlyenes, as shown in Scheme III. The acetateprotecting groups of 19a-e are removed with LiOH in methanol to affordthe corresponding 4-substituted-[1,2,3]-triazoles 20a-e.

Substitution of the 2-position hydrogen with a fluorine is accomplishedby electrophilic fluorination of 9b (Scheme IV) using Selectfluor®. Theazido group of intermediate 22 is converted to a series of4-substituted-[1,2,3]-triazoles 23a-b via the standard conditions.

In another embodiment, the following compounds are described:

Minimum inhibitory concentration (μg/mL)^(a) S. aureus 96:11480 S.pneumoniae H. influenzae 29213 Ery-R 49619 163 303 49247 Entry R n Ery-S(MLSb) Ery-S Ery-R (MefA) Ery-R (ermB) Ery-S TEL ≦0.125 ≦0.125 ≦0.125≦0.125 ≦0.125 4 AZI ≦0.125 <64 ≦0.125 >64 >64 2 11a 11b 11c

3 4 5 1 ≦0.125 ≦0.125 1 0.25 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125≦0.125 >64 2 0.25 >64 8 16 12a 12b 12c

3 4 5 0.25 ≦0.125 ≦0.125 0.5 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125≦0.125 ≦0.125 8 8 1 64 8 16 13a 13b 13c

3 4 5 1 0.25 0.5 2 0.25 1 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 0.5 16 82 >64 8 64 14a 14b 14c

3 4 5 2 ≦0.125 ≦0.125 2 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 0.5 ≦0.125≦0.125 >64 ≦0.125 0.25 >64 4 64 15a 15b 15c

3 4 5 2 ≦0.125 ≦0.125 2 4 0.25 ≦0.125 ≦0.125 ≦0.125 1 2 0.25 >64 644 >64 64 16 16a 16b 16c

3 4 5 0.5 0.125 ≦0.125 nt ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125≦0.125 ≦0.125 >64 ≦0.125 0.25 16 2 8 17a 17b 17c

3 4 5 1 ≦0.125 0.25 1 ≦0.125 0.5 ≦0.125 ≦0.12  ≦0.125 ≦0.125 ≦0.12≦0.125 >64 1 2 >64 16 32 18a 18b 18c

3 4 5 1 1 ≦0.125 2 2 ≦0.125 ≦0.125 ≦0.125 ≦0.125 0.5 4 ≦0.125 >64 6464 >64 32 8 ^(a)National Committee for Clinical Laboratory Standards.Methods for Dilution Antimicrobial Susceptibility Tests for Bacteriathat Grow Aerobically, 6^(th) ed.; Approved standard: NCCLS DocumentM7-A6, 2003.

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae H. influenzae 25923 49619 163 303 49247 Entry REry-S RN220 Ery-S Ery-R (MefA) Ery-R (ermB) Ery-S TEL ≦0.25 2 ≦0.125≦0.125 ≦0.125 4 20a

0.25 8 ≦0.0625 0.125 2 NT 20b

0.25 8 ≦0.0625 ≦0.06 1 NT 20c

1 8 ≦0.0625 0.5 2 NT 20d

1 8 ≦0.0625 0.5 2 NT 20e

≦0.25 8 ≦0.0625 0.5 2 NT

In another embodiment, the following compounds are described:

S. aureus S. pneumoniae 96:11480 Ery 163 303 H. influenzae 29213 R 49619Ery-R Ery-R 49247 Entry R Ery-S (MLSb) Ery-S (MefA) (ermB) Ery-S TEL≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 4 AZI ND ≦0.125 >64 ≦0.125 >64 >6423a

≦0.125 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2 23b (CEM- 101)

≦0.06 ≦0.125 ≦0.125 ≦0.125 ≦0.125 2

In each of the foregoing embodiments, the primary screening panelconsisted of relevant Staph. aureus, S. pyogenes, S. pneumoniae(including strains resistant to azithromycin and telithromycin). MICsagainst all pathogens were determined using broth microdilution methodas per NCCLS guidelines. Compounds described herein, such as CEM-101were found to be highly potent having MICs against S. pneumoniae (3773)of ≦0.125 μg/mL and S. pyogenes (1850) of 0.5 μg/mL, compared to 1 and 8μg/mL, respectively for Telithromycin. CEM-103 (20c), an analogue ofCEM-101 that contains the 3-O-cladinose was found to be less active.Non-heteroaromatic substituted triazole containing ketolides were lessactive.

The ketolides were tested against erythromycin-sensitive (Ery-S) anderythromycin-resistant (Ery-R) strains of S. aureus (29213 (Ery-S) and96:11480 (Ery-R)), S. pneumoniae (49619 (Ery-S) and 163 and 303 (Ery-R))and H. influenzae (49247 (Ery-S)) (Tables 1-3). The broth micro-dilutionmethod was used to determine the Minimum Inhibitory Concentrations(MICs) against all pathogens as per the Clinical and LaboratoryStandards Institute (CLSI).

The chain length of the alkyl side chain had a affected activity (Table1). For example, the 3-carbon linked phenyl substituted triazole 11a wasless active against Ery-S and Ery-R S. aureus and was inactive againstEry-R S. pneumoniae 303 (ermB) a the tested concentrations, whereas thecorresponding 4- and the 5-carbon linked phenyl substituted triazoles11b and 11c were more active against these organisms. A similar trendwas observed for the 2-pyridyl substituted triazoles 14a-c, the3-amino-phenyl substituted triazoles 16a-c, and the 2,5-dichlorophenoxysubstituted triazoles 17a-c.

The 4-carbon linked 2-pyridyl substituted triazole 14b and the3-amino-phenyl substituted triazole 16b possessed the highest potencyagainst S. pneumoniae 303, both having MIC values (≦0.125 μg/mL)comparable to telithromycin. The ketolide containing the 4-carbon linked3-pyridyl substituted triazole 15b was less active against this strain(MIC of 64 μg/mL). Within this series antibacterial activity wasimproved by extending the carbon linker to 5 atoms, for example the MICagainst S. pneumoniae 303 for compound 15c improved from 64 to 4 μg/mL.A similar effect was also observed for the benzo-triazole containingketolide 18c against S. aureus but 18c was still inactive against S.pneumoniae 303. It is appreciated that a balance between the length ofthe linker and nature of the aromatic substitution of the triazole mayaffect the overall activity against macrolide resistant S. pneumonia andS. aureus.

A correlation between linker length and activity was also observed forH. influenzae (49247) where the most potent ketolide series had thesubstituted triazole linked through either a 4-carbon (11b-14b, 16b,17b) or a 5-carbon (15c, 18c) chain. Interestingly, the most potentaromatic series against H. influenzae was the 3-amino-phenyl with a 3-,4- or 5-carbon linker (16a, 16b, 16c) having MICs of 16, 2, and 8 μg/mL,respectively,

The macrolides containing a cladinose at the 3 position were all highlyactive against Ery-S S. pneumoniae (49619) (Table 2). However, theseanalogs were less potent than telithromycin against Ery-R strains. TheMICs were significantly higher for the cladinose containing analogs witheither 2-pyridyl, 2-aminophenyl or 2,6-dichlorophenyl triazolesubstituents than for the corresponding ketolides (20a, 20c, and 20dversus 14b, 16b, and 17b). Conversely, antibacterial activity wasre-established for ketolide analogs 15b (3-pyridyl) and 18b(benzo-triazole) by replacing the keto with the cladinose group inanalogs 20b (3-pyridyl) and 20e (benzo-triazole). The MICs improved from64 μg/mL for 15b and 18b to 1 and 2 μg/mL for 20b and 20e, respectively.A similar activity trend was also observed for Ery-R S. pneumoniae 163(MefA).

COMPOUND EXAMPLES

A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-(3-dimethylamine-4-deoxy-6-O-acetyl-glu-copyranosyl)-2-fluoro-3-oxo-erythronolideA, 11,12-carbamate (15 mg, 0.019 mmol), 6-Ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 14 mg of the desired compound. MS:C₄₄H₆₆FN₇O₁₂ calculated M⁺=903.5. Found: M+H⁺=904.5.

11-N-4-(3-aminophenyl)-[1,2,3]triazol-1-yl]-butyl-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate (CEM-101). A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (17 mg, 0.023 mmol), 3-Ethynyl-phenylamine (5.4 mg,0.046 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heated to70° C. After 16 h, the mixture was concentrated and directly subjectedto silica gel chromatography (9:1, chloroform:methanol plus 1% ammoniumhydroxide) to give 17 mg of the desired compound, MS C₄₃H₆₅FN₆O₁₀calculated M⁺=844.47. Found: M+H⁺=845.5.

11-N-{4-[4-(6-Amino-pyridin-2-yl)-[1,2,3]triazol-1-yl]-butyl}-5-desosaminyl-3-oxo-2-fluoro-erythronolideA,-11,12-cyclic carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-desosamynyl-3-oxo-2-fluoro-erythronolideA, 11,12-carbamate (15 mg, 0.02 mmol), 6-ethynyl-pyridin-2-ylamine (4.7mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 70° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 14 mg of the desired compound OP1357. MS:C₄₂H₆₄FN₇O₁₀ calculated M⁺=845.5. Found: M+H⁺=846.5.

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-O-dasosaminyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-Azido-butyl)-6-O-methyl-5-O-desosaminyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.0039 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₀ calculated M⁺=866.5. Found: M+H⁺867.5.

11-N-[4-(4-Benzotriazol-1-ylmethyl-[1,2,3]triazol-1-yl)-butyl]-6-O-methyl-5-mycaminosyl-3-oxo-erythronolideA, 11,12-carbamate. A mixture of11-N-(4-azido-butyl)-6-O-methyl-5-mycaminosyl-3-oxo-erythronolide A,11,12-carbamate (3 mg, 0.004 mmol), 1-Prop-2-ynyl-1H-benzotriazole (3mg, 0.4 mmol), Cul (1 mg, 0.005 mmol), and toluene (0.2 mL) was heatedto 80° C. After 16 h, the mixture was concentrated and directlysubjected to silica gel chromatography (9:1, chloroform:methanol plus 1%ammonium hydroxide) to give 3 mg of the desired compound. MS:C₄₄H₆₆N₈O₁₁ calculated M⁺=882.5. Found: M+H⁺=883.5.

METHOD EXAMPLES

SAL (20 strains, representing 11 serotypes) and Shigella (40; fourspecies) were tested by CLSI broth microdilution methods with M100-S18breakpoints applied. C. jejuni (20) and H. pylori (23) were tested byMueller-Hinton agar dilution method, supplemented with sheep blood, andC. jejuni results were confirmed by Etest (AB BIODISK, Solna, Sweden).Key comparison agents were tested: AZI, CLR, TEL, levofloxacin (LEV),amoxicillin/clavulanate (A/C) and trimethoprim/sulfamethoxazole(TMP/SMX).

CEM-101 demonstrated activity against food-borne GDPs Salmonella (MIC50,4 μg/ml), Shigella (MIC50, 8 μg/ml) and C. jejuni (MIC50, 1 μg/ml). Thiswas comparable or superior (MIC50 ranges) to: TEL (8-16 μg/ml), ERY(2->4 μg/ml), AZI (4 μg/ml) and A/C (2-8 μg/ml). CLR results werediverse (MIC50 range 0.015->16 μg/ml) as well as were TMP/SMX; LEV wasmost active (MIC50, ≦0.12 μg/ml). HP CEM-101 MIC results were groupedfrom 0.03-0.25 μg/ml and at 2 or 4 μg/ml; the latter corresponding toCLA-R (>16 μg/ml) strains.

CEM-101 Comparator (Drug) a

Comparator CEM-101 (drug)^((a)) Organism (no.) 50% 90% Range 50% 90%Range C. jejuni (20) 1 4 1-8  2 4 1-8 (CLA) H. pylori (23) 0.06 0.250.03-4    0.03 0.12 ≦0.015->16 (CLA) Salmonella spp. (20) 4 >16 1->16 48 2-8 (AZ) Shigella spp. (40) 8 16 1->16 4 8 1->16 (AZ) ^((a))Comparatordrug in parentheses (AZI [AZ] or CLR [CLA]).

Some special organism subsets were specifically tested includingCampylobacter jejuni, Helicobacter pylori and Enterobacteriaceae(Salmonella spp., Shigella spp.). Reference-quality methods were appliedincluding those of the Clinical and Laboratory Standards Institute(CLSI) and the alternative Etest (AB Biodisk, Solna, Sweden) method. Inrecent years, MLSB-ketolide class compounds have been used for a numberof gastrointestinal (GI) infections and resistances to several potentialtreatment agents requires a search for novel therapeutic options. Thecompounds described herein were screened in vitro for potentialapplication for these GI indications.

MATERIALS AND METHODS. Susceptibility testing methods: For C. jejuni, N.gonorrhoeae and H. pylori, CLSI M7-A7 (2006) and M100-S18 (2008) agardilution methods were used as follows: Mueller-Hinton (MH) agar with 5%sheep blood for H. pylori and Campylobacter spp. 10⁵ CFU/spot inocula.Endpoints read at 24 (C. jejuni) or 72 hours (H. pylori). Appliedincubation environments appropriate for species (added CO2 ormicroaerophilic).

96-well frozen-form assay panels were also used, produced by JMILaboratories and consisted of cation-adjusted MH broth for testing theEnterobacteriaceae. Comparator agents were tested

Table 1 summarizes CEM-101 activity against H. pylori. Eight strainswere compared by testing five drugs, including CEM-101. Results showedthat CEM-101 was slightly less active than CLR or aminopenicillins(MIC50, ≦0.015 μg/ml); however the comparator activity measurements wereEtest results, not the reference agar dilution method. Inter-method datafor CLR (data not shown) exhibited a trend toward lower Etest results(four-fold). CEM-101 MICs for the CLR-resistant (>16 μg/ml) strains wereonly 2 or 4 μg/ml.

TABLE 1 Comparative activity of CEM-101 tested against 103 isolates ofenteritis- producing pathogens showing MIC (μg/ml) % by category^((a)).Organism susceptible/ (no. tested), 50% 90% Range resistant Salmonellaspp. (20)^((b)) CEM-101 4 >16    1->16 —/— TEL 8 >16 0.015->16 —/—Erythromycin >4 >4 0.25->4 —/— CLR 0.015 >16 0.015->16 —/— AZI 4 8  2-8—/— Clindamycin >4 >4 0.25->4 —/— Quinupristin- >4 >4 0.25->4 —/—dalfopristin Amoxicillin- 2 8  0.5->8 95.0/0.0  clavulanate Cefdinir0.25 0.5 ≦0.12-0.5   100.00/0.0   Levofloxacin ≦0.12 1 ≦0.12-4  95.0/0.0  Trim-sulfa^((g)) ≦0.25 ≦0.25 ≦0.25 100.00/0.0   Shigella spp.(40)^((c)) CEM-101 8 16    1->16 —/— TEL 16 16    2->16 —/—Erythromycin >4 >4 0.25->4 —/— CLR >16 >16 0.015->16 —/— AZI 4 8   1->16 —/— Clindamycin >4 >4 0.25->4 —/— Quinupristin- >4 >4 >4  —/—dalfopristin Amoxicillin- 8 >8   2->8 72.5/0.0  clavulanate Cefdinir0.25 0.25 ≦0.12-0.5   100.0/0.0  Levofloxacin ≦0.12 ≦0.12 ≦0.12-0.25 100.0/0.0  Trim-sulfa^((g)) >4 >4 ≦0.25->4  37.5/62.5 C. jejuni (20)CEM-101^((d)) 1 4  1-8 —/— CLR^((e)) 2 4  1-8 —/— Ciprofloxacin^((e))0.25 >32  0.03->32 —/— Erythromycin^((e)) 2 4 0.5-4 —/—Tetracycline^((e)) 64 >256  0.06-256 —/— H. pylori (23/8)^((f)) CEM-1010.06 0.25 0.03-4  —/— CLR 0.03 0.12 ≦0.015->16  91.3/8.7  Ampicillin≦0.015 — ≦0.015-0.03   —/— Metronidazole 0.5 — 0.06-64 —/— Tetracycline0.06 — ≦0.015-0.25   —/— ^((a))Criteria as published by the CLSI [2008].— = no interpretational criteria have been established. ^((b))Includes:Salmonella dublin (1 strain), S. enteritidis (4 strains), S. hadar (1strain), S. heidelberg (1 strain), S. infantis (1 strain), S. paratyphi(3 strains), S. typhi (3 strains), S. typhimurium (1 strain), Group BSalmonella (2 strains), Group C Salmonella (1 strain), and Group DSalmonella (2 strains). ^((c))Includes: Shigella boydii (6 strains),dysenteriae (3 strains), S. flexneri (14 strains), and S. sonnei (17strains). ^((d))Tested using the agar dilution method recommended by theCLSI (M7-A7). ^((e))Tested by Etest using manufacturer's recommendations(AB BIODISK, Solna, Sweden). ^((f))Twenty-three were tested by CLSI(2006) method and eight by Etest; ampicillin, metronidazole andtetracycline results were produced by Etest.^((g))Trimethoprim-sulfamethoxazole

All organisms to be tested were collected from patients in USA andEuropean medical centers from 2005 to present. Sources of recoveredisolates included bloodstream, skin and soft tissue, respiratory tractinfections and gastrointestinal tract. Unusual/rare organism species andphenotypes required use of strains isolated prior to 2005 or from othergeographic areas. Organisms were tested: H. pylori (23; twoCLR-resistant), C. jejuni (20; fluoroquinolone andtetracycline-resistant samples), Salmonella spp. (20; 11 groups),Shigella spp. (40; four species).

Table 2 shows the CEM-101 MIC distributions for all tested strains (fourspecies; 103 strains). CEM-101 MIC results for the H. pylori were lowest(≦0.03-0.4 μg/ml), while MICs for the Enterobacteriaceae could range upto ≧16 μg/ml.

TABLE 2 CEM-101 MIC distributions for all tested populations ofpathogens in this protocol (103 strains). Organism (no. tested) ≦0.030.06 0.12 0.25 0.5 1 2 4 8 ≧16 H. pylori (23) 1 15 2 3 0 0 1 1 0 0 C.jejuni (20) 0 0 0 0 0 10 0 8 2 0 Salmonella spp. (20) 0 0 0 0 0 3 4 4 45 Shigella spp. (40) 0 0 0 0 0 2 0 14 18 6

CEM-101 exhibits potent activity against staphylococci (MIC50, 0.06μg/ml), streptococci (MIC50, 0.015 μg/ml), enterococci (MIC50/90, 0.25μg/ml) and other Gram-positive cocci including strains resistant to ERYand CLN, by Etest using manufacturer's package insert directions (ABBIODISK). CEM-101 and 14 selected comparison antimicrobial agents weretested.

Quality control (QC) ranges and interpretive criteria for comparatorcompounds were as published in CLSI M100-S18 (2008); tested QC strainsincludes S. aureus ATCC 29213, E. faecalis ATCC 29212, S. pneumoniaeATCC 49619, H. pylori ATCC 43504, and C. jejuni ATCC 33560.

CEM-101 inhibited H. pylori (MIC50, 0.06 μg/ml), and various othergastrointestinal pathogens. CEM-101 activity against H. pylori (MIC90,0.25 μg/ml) was most like that of CLR (MIC90, 0.12 μg/ml). CEM-101 wasalso most like other macrolides versus C. jejuni (MIC50 and MIC90results, 1-4 μg/ml). CEM-101 also showed promise for application againstintestinal infections caused by Salmonella spp. and Shigella spp., anactivity similar to that of AZI.

EXAMPLE. Animal Model of H. pylori Gastroenteritis. Female C57BL/6 mice(age, >7 weeks) are inoculated with the SS1 strain of H. pylori (Lee, etal., 1997, Gastroenterology 112:1386-1397) via gavages of 100-μlsuspensions (109 CFU/ml). Infection is monitored (after Crone, et al.,Clin Diagn Lab Immunol. 2004 July; 11(4): 799-800) by analyzing fecalpellets using a monoclonal antibody-based enzyme-linked immunosorbentassay (FemtoLab H. pylori Cnx; Connex, Martinsried, Germany) to detectinfection by H. pylori (SS1). According to manufacturer's guidelines, anoptical density (OD) of <0.150 was defined as negative for H. pylori,and an OD of >0.150 was considered a positive test result.

Infection level and level of gastritis present are also measured usinghistological methods and culture of tissue homogenates. Mice aresacrificed by CO₂ asphyxiation and cervical dislocation, after which thestomachs are excised for histological examination and bacterial culture.Paraffin-embedded sections are stained with hematoxylin and eosin forhistology and with a modified May-Grünwald-Giemsa stain to assessbacterial colonization (Laine, et al., 1997, Gastrointest. Endosc.45:463-467). Gastritis is assessed in the body and the antrum by using amodified Sydney grading system for gastritis (Lee, et al., 1997). Theseverity of gastritis and bacterial colonization density are assessedblindly by an impartial observer.

CEM-101 is administered with and without a proton pump inhibitorcompound according to the dosage regimen in the protocol and the degreeof infection monitored as described above.

EXAMPLE. Human THP-1 macrophages were used. Accumulation was measured bymicrobiological assay. Intracellular activity was determined againstphagocytized S. aureus (ATCC 25923; MICs: CEM-101, 0.125 mg/L; AZI, 0.5mg/L) using a dose-response approach (AAC 2006; 50:841-51). Verapamil(100 μM) and gemfibrozil (250 μM) were used as inhibitors ofP-glycoprotein and MRP, respectively (AAC, 2007; 51:2748-57).

Accumulations and activities after 24 h incubation, with and withoutefflux transporters inhibitors, are shown in the following Table, whereCc/Ce is the apparent cellular to extracellular concentration ratio, andE_(max) is the maximal decrease of intracellular cfu compared topost-phagocytosis inoculum (calculated from non-linear regression[sigmoidal] of dose-effect response experiments).

AZI CEM-101 Intracellular activity Intracellular activity (Δ log cfu at24 h) (Δ log cfu at 24 h) Static dose Cc/Ce¹ Static dose ConditionCc/Ce¹ (24 h) (mg/L) E_(max) ² (24 h) (mg/L) E_(max) ² control 127.7 ±23.5 ~7.0  0.10 ± 0.09 268.1 ± 7.1  ~0.02 −0.85 ± 0.23^((b)) Verapamil  216.37 ± 46.6^((a)) ~0.2 −0.37 ± 0.15 290.2 ± 12.9 ~0.03 −0.59 ±0.22^((b)) Gemfibrozil 129.12 ± 2.69  ~3.8 −0.12 ± 0.20 308.2 ± 47.8~0.03 −0.73 ± 0.20^((b)) ^((a))Statistically significant from bothcontrol and Gemfibrozil; ^((b))not statistically significant.

EXAMPLE. Intracellular activity of antibiotics. The determination ofantibiotic activity against intraphagocytic S. aureus strain ATCC 25923was determined. Full dose-responses studies were performed to assess theimpact of active efflux in the modulation of the intracellular activityof CEM-101 and AZI against intraphagocytic S. aureus (strain ATCC 25923[MICs: CEM-101, 0.125 mg/L; AZI, 0.5 mg/L]. Antibiotics were compared at24 h for: (i) their relative static concentration (Cs), and (ii) theirrelative maximal efficacy (E). While verapamil (but not gemfibrozil)increases the intracellular activity of AZI, neither inhibitor havesignificant effect on the activity of CEM-101, suggesting that thelatter, in contrast with AZI, is not a substrate of the correspondingeukaryotic transporters.

EXAMPLE. Cellular accumulation of antibiotics. The cellular content inmacrolides was measured in THP-1 macrophages by microbiological assay,using S. aureus ATCC 25923 as test organism. Cell proteins was assayedin parallel using the Folin-Ciocalteu/Biuret method. The cell associatedcontent in macrolides was expressed by reference to the total cellprotein content, and converted into apparent concentrations using aconversion factor of 5 μL per mg of cell protein (as commonly used forcultured cells).

The cellular accumulation of CEM-101 in comparison with that of AZI inTHP-1 cells was first measured FIG. 5 (panel A). At 24 h, bothantibiotics concentrate to large extents in cells, but with a largervalue (Cc/Ce) for CEM-101. In a second stage, whether CEM-101 is asubstrate of Pgp or MRP efflux transporters was investigated FIG. 5(panel B). Using a Pgp (verapamil) or MRPs inhibitor (gemfibrozil), nosignificant variations of the cellular accumulation of CEM-101 areobserved while verapamil increases significantly the cellularaccumulation of AZI.

Uptake of CEM-101 was linear over time, reaching accumulation levelsabout 375-fold within 24 h (AZI, 160X, CLR, 30X, TEL, 21X). Accumulationwas suppressed by acid pH or addition of the proton ionophore monensin,but not modified by verapamil or gemfibrozil (preferential inhibitors ofPgp and MRP, respectively). Panel B shows that the accumulation of bothCEM-101 and AZI was reduced when the experiments were conducted atacidic pH, with the change occurring almost entirely when the pH wasbrought from 7 to 6. Panel C shows that monensin, which is known todecrease the cellular accumulation of many weak organic bases, alsoalmost completely suppressed the accumulation of both CEM-101 and AZI.In contrast, verapamil, an inhibitor of the P-glycoprotein effluxtransporter (Pgp, also known as MDR1), increased the accumulation of AZIwithout affecting that of CEM-101, whereas gemfibrozil, an inhibitor ofmultidrug resistance proteins (MRP) and other organic anion transportersdid not affect either compound. Neither verapamil nor gemfibrozilaffected the accumulation of TEL or CLR (data not shown). The efflux ofCEM-101 from cells incubated with 10 mg/L of CEM-101 for 1 h and thentransferred into drug-free medium was examined. Efflux proceeded in abimodal fashion, with half of the cell-associated drug being releasedwithin approximately 10 min, followed by a slower release phase ofseveral hours (data not shown).

EXAMPLE. Macrolides accumulate in eukaryotic cells and are consideredadvantageous for the treatment of intracellular infections. Ketolidesare active against erythromycin-resistant organisms. The cellularaccumulation and intracellular activity of CEM-101 towards theintracellular forms of Staphylococcus aureus (S. a.), Listeriamonocytogenes (L. m.), and Legionella pneumophila (L. p.) in comparisonwith AZI, CLR, and TEL is shown in the following table.

MIC^(a) Cs^(b) E_(max) ^(c) CEM-101 S.a. 0.06 0.022 −0.86 L.m. 0.0040.11 −0.66 L.p. 0.004 0.018 −1.03 AZI S.a. 0.5 >50 0.04 L.m. 1 11.6−0.81 L.p. 0.016 2.90 −0.83 CLR S.a. 0.5 0.84 −0.18 L.m. L.p. 0.007 0.12−0.71 TEL S.a. 0.25 0.63 −0.29 L.m. L.p. 0.007 0.06 −0.63 ^(a)mg/L;^(b)static concentration (mg/L) at 24 h; ^(c) Δ log₁₀ CFU at 24 hcompared to the post-phagocytosis inoculum

EXAMPLE. MICs and extracellular activities of antibiotics weredetermined in MHB at both neutral and acidic pH. Intracellular activitywas determined against S. aureus (ATCC 25923) phagocytosed by THP-1macrophages as previously described (AAC, 2006, 50:841-851). Resultswere expressed as a change of efficacy compared to time 0 h.

Conditions CEM-101 AZI CLR TEL MICs (mg/L) (i) pH 7.4 0.125 0.5 0.5 0.5(ii) pH 5.5 1-2 256 16 8 Extracellular activity (24 h): Δ log cfu fromtime 0 h (i) Broth pH 7.4 Emax¹ −1.4 ± 0.1 −1.2 ± 0.6 −1.4 ± 0.2 −1.0 ±0.4 Static ~0.06 ~3.63 ~1.41 ~0.28 dose² R² 0.964 0.860 0.965 0.868 (ii)Broth pH 5.5 Emax¹ −1.6 ± 0.4 +2.1 ± 0.1 −1.5 ± 0.8 −1.4 ± 0.9 Static~1.48 / ~10.47 ~9.33 dose² R² 0.915 / 0.911 0.879 Intracellular activity(24 h): Δ log cfu from time 0 h Emax¹ −0.8 ± 0.2 0.10 ± 0.0 −0.1 ± 0.1−0.4 ± 0.2 Static ~0.02 ~7.8 ~0.98 ~0.23 dose² R² 0.906 0.980 0.9740.935 THP-1 Emax¹ −0.8 ± 0.2  0.1 ± 0.1 −0.1 ± 0.1 −0.4 ± 0.1 Static~0.02 ~10 ~0.98 ~0.28 dose² ¹Maximal decrease of intracellular cfucompared to initial, post-phagocytosis inoculum (calculated fromnon-linear regression [sigmoidal] of dose-effect response) run in broth(extracell.) or with infected macrophages (intracell.) ²Extracellularconcentration (Cs in mg/L) yielding an apparent static effect.Comparative pharmacological descriptors (Emax and static concentrations[Cs]) obtained from the dose-responses studies. Dose-response studies inMueller-Hinton broth. Against S. aureus ATCC 25923 and in broth, at pH7.4, CEM-101 is systematically more active than AZI, CLR and TEL; at pH5.5, AZI, CLR and TEL show significant decrease of their potencies,while CEM-101 shows less change.Compared to AZI, CLR and TEL, CEM-101 activity was less affected byacidic pH of the broth and showed greater potency (lower static dose)and larger maximal efficacy (Emax) against intracellular S. aureus.

EXAMPLE. Cell lines. Experiments were performed with THP-1 cells (ATCCTIB-202; American Tissue Culture Collection, Manassas, Va.), a humanmyelomonocytic cell line displaying macrophage-like activity (see, e.g.,Barcia-Macay et al., Antimicrob. Agents Chemother. 50:841-851 (2006)).Assay of the cell-associated macrolides and calculation of the apparentcellular- to extracellular-concentration ratios. Macrolides were assayedby a microbiological method, using S. aureus ATCC 25923 as a testorganism. Cell proteins were measured in parallel using theFolin-Ciocalteu/biuret method. The cell-associated contents inmacrolides were expressed by reference to the total cell protein contentand converted into apparent concentrations using a conversion factor of5 μL per mg of cell protein, an average value found for many culturedcells.

Bacterial strains, susceptibility testing, and 24-h dose-response curvestudies with broth. S. aureus ATCC 25923 (methicillin [meticillin]sensitive), L. monocytogenes strain EGD, and L. pneumophila strain ATCC33153 were used in the present study. MIC determinations were performedin Mueller-Hinton broth (for S. aureus) and tryptic soy broth (for L.monocytogenes) after a 24-h incubation, or in a-ketoglutarate-bufferedyeast extract broth (for L. pneumophila) after a 48-h incubation. For S.aureus studies, 24-h concentration-response experiments in acellularmedium were performed in Mueller-Hinton broth.

Cell infection and assessment of antibiotic intracellular activities.Infection of THP-1 cells and assessment of the intracellular activity ofantibiotics were performed using conventional methods for S. aureus andL. monocytogenes or with minor adaptations for L. pneumophila using (i)a multiplicity of infection of 10 bacteria per macrophage and (ii)gentamicin (50 mg/liter) for 30 to 45 min for the elimination ofnonphagocytosed bacteria.

Statistical analyses. Curve-fitting statistical analyses were performedwith GraphPad Prism version 4.03 and GraphPad Instat version 3.06(GraphPad Software, San Diego, Calif.).

EXAMPLE. Susceptibility toward S. aureus ATCC 25923, Listeriamonocytogenes EGD, and Legionella pneumophila ATCC 33153. CEM-101 showedlower MICs than AZI against the three selected organisms (S. aureus,0.06 and 0.5 mg/liter; L. monocytogenes, 0.004 and 1 mg/liter; and L.pneumophila, 0.004 and 0.016 mg/liter) in conventional susceptibilitytesting. The MICs of CEM-101, TEL, AZI, and CLR against S. aureus and L.monocytogenes were measured in broths adjusted to pH values ranging from5.5 to 7.4. The range was selected to cover the values at which theantibiotics could be exposed in the extracellular milieu orintracellularly for the two organisms considered. As illustrated in FIG.1, all four drugs showed a marked decrease in potency against bothorganisms when the pH was decreased from 7.4 to 5.5, with AZIdemonstrating the most significant loss of activity. CEM-101 retainedthe most activity, consistently showing the lowest MICs throughout theentire pH range investigated, with values (mg/liter) ranging from 0.06(pH 7.4) to 0.5 (pH 5.5) for S. aureus (ATCC 25923) and 0.0039 (pH 7.4)to 0.25 (pH 5.5) for L. monocytogenes (EDG). For L. pneumophila (datanot shown), the MIC of CEM-101 increased from 0.005 to 0.01 and that ofAZI from approximately 0.01 to 0.25 mg/liter when the pH of the brothwas decreased from 7.4 to 6.5 (no determination could be made at lowerpH values because of absence of growth).

EXAMPLE. Time and concentration effects against extracellular andintraphagocytic S. aureus. Short-term (6-h) time-kill curves wereobtained for CEM-101 in comparison with those for AZI against S. aureus(ATCC 25923) in broth and after phagocytosis by THP-1 macrophages usingtwo single fixed concentrations of 0.7 and 4 mg/liter. The lowerconcentration was chosen to be relevant to the serum concentration ofAZI and CEM-101, and the higher concentration was selected to be abovethe MIC of AZI for the organisms of interest. Results presented in FIG.3 show that under these conditions, only CEM-101 was able tosignificantly decrease CFU in broth as well as in THP-1 macrophages atthe 0.7-mg/liter concentration. At the 4-mg/liter concentration inbroth, AZI eventually achieved the same antibacterial effect as CEM-101,but at a lower rate (5 h compared to 1 h). In THP-1 macrophages, noconsistent activity was detected for AZI, even at the 4-mg/literconcentration, whereas CEM-101 again achieved a reduction ofapproximately 1.5 log 10 CFU, similar to the magnitude seen at the0.7-mg/liter concentration. In all situations with CEM-101, the maximaldecrease of CFU was obtained within 1 h and was maintained thereafter.

We then performed concentration-response experiments at a fixed timepoint (24 h) to obtain the pertinent pharmacological descriptors ofCEM-101 activity (relative potency [50% effective concentration {EC50}],apparent static concentration [C_(s)], and relative maximal efficacy[E_(max)] in comparison with CLR, AZI and TEL activity (additionaldetails are described in Barcia-Macay et al., Pharmacodynamic evaluationof the intracellular activities of antibiotics against Staphylococcusaureus in a model of THP-1 macrophages Antimicrob. Agents Chemother.50:841-851 (2006)). Data are presented in FIG. 2 as a function of (i)weight concentrations (mg/liter) and (ii) multiples of the MICs (asdetermined in broth at pH 7.4). The numerical values of thecorresponding pharmacological descriptors are shown in the Table.Pertinent regression parameters^(a) (with confidence intervals [CI]),and statistical analysis of the dose-response curves illustrated in FIG.2.

broth ⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄) (Cl) C_(S) ^(⋄⋄) R²CEM-101 −1.37 mg/L 0.03 0.06 0.973 (−1.67 to −1.08) (0.02 to 0.06) a; Aa; A xMIC 0.48 0.88 (0.26 to 0.91) a; A TEL −1.00 mg/L 0.12 0.29 0.892(−1.78 to −0.22) (0.03 to 0.52) a; A b; A x MIC 0.46 0.96 (0.11 to 2.06)a; A AZI −1.23 mg/L 1.78 3.4 0.872 (−2.55 to 0.083) (0.45 to 7.02) a; Ac; A x MIC 3.55 6.87 (0.90 to 14.0) b; A CLR −1.41 mg/L 0.80 1.32 0.956(−1.95 to −0.87) (0.41 to 1.56) a; A c; A x MIC 1.59 2.65 (0.81 to 3.1) a, b; A THP-1 macrophages ⁺⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄) (Cl)C₅ ^(⋄⋄) R² (CI) CEM-101 −0.86 mg/L  0.0068 0.022 0.927  (−1.36 to−0.37) (0.0023 to 0.020) a; B a; B x MIC 0.11  0.35 (0.037 to 0.32) a; BTEL −0.29 mg/L 0.024 0.63 0.954 (−0.70 to 0.12)  (0.007 to 0.088) b; Bb; B x MIC 0.097 1.04 0.027 to 0.35 a; B AZI  0.04 mg/L 0.11  >50 0.983(−0.23 to 0.32)  (0.05 to 0.22) b; B c; B x MIC 0.22  >100  0.11 to 0.45a; B CLR −0.18 mg/L 0.046 0.84 0.974 (−0.52 to 0.16) (0.018 to 0.12) b;B b, c; B x MIC 0.093 1.68 0.035 to 0.25 a; B ^(a) using all data pointsshown in FIG. 4 (data from samples without antibiotic when theextracellular concentration of an antibiotic is lower than 0.01 x MIC(5) ⁺ original inoculum [time = 0 h]: 0.97 ± 0.24 × 10⁶ CFU/mL (n = 3)⁺⁺ original (post-phagocytosis) inoculum [time = 0 h]: 2.74 ± 0.55 × 10⁶CFU/mg protein (n = 3) ^(♦)CFU decrease (in log₁₀ units) at time = 24 hfrom the corresponding original inoculum, as extrapolated for antibioticconcentration = ∞; samples yielding less than 5 counts were consideredbelow detection level. ^(⋄)concentration (in mg/L or in x MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1); ^(⋄⋄)concentration (in mg/L or in x MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation; StatisticalAnalyses. Analysis of the differences between antibiotics (per columnfor the corresponding rows; one-way ANOVA with Tuckey test for multiplecomparisons between each parameter for all drugs): figures withdifferent lower case letters are significantly different from each other(p < 0.05). Analysis of the differences between broth and THP-1macrophages (per row for the corresponding columns; unpaired, two-tailedt-test): figures with different upper case letters are significantlydifferent from each other (p < 0.05).

The activities in both broth and THP-1 macrophages developed in aconcentration-dependent fashion, as denoted by the sigmoidal shape ofeach best-fit function (Hill equation). In broth, the relative efficacyof CEM-101 (E_(max) of −1.37 log₁₀) was similar to that of the otherdrugs (E_(max) values of −1.00 to −1.41 log₁₀). In THP-1 macrophages,the relative efficacy of CEM-101 was significantly decreased compared tothat in broth (E_(max) of −0.86 log₁₀), but not to the same extent asthose of the other drugs, which essentially became bacteriostatic only(E_(max) values of 0.04 to −0.29 log₁₀). On a weight basis, CEM-101 hadhigher relative potencies (lower E₅₀ values) and lower staticconcentrations (lower C_(s) values) than all three comparator drugs inboth broth and in THP-1 macrophages. When the data were analyzed as afunction of equipotent concentration (multiples of the MIC), thesedifferences in EC₅₀ values were reduced, indicating that the MIC was themain driving parameter in this context. In broth, even when analyzed asmultiples of the MIC, CEM-101 and CLR still showed significantly lowerEC₅₀s than TEL and AZI.

Example. Activity against intraphagoctic L. monocytogenes and L.pneumophila. The same approach was used as that for S. aureus to assessthe activities of CEM-101 and AZI against phagocytized L. monocytogenesand L. pneumophila to obtain information on concentration-effectrelationships and on the corresponding pertinent pharmacologicaldescriptors. As shown in FIG. 4, a relationship compatible with the Hillequation was observed in all cases, although the limited growth of L.pneumophila made the fitting of functions somewhat more uncertain. Whenthe data were plotted against weight concentration, it appeared thatCEM-101 had a higher relative potency (lower EC50) than AZI for both L.monocytogenes and L. pneumophila. This difference was reduced butnevertheless remained significant when data for L. pneumophila wereplotted against multiples of the MIC, indicating that the MIC was animportant but not the exclusive driver of intracellular activity againstthis organism. Conversely, no difference in the responses was seen forL. monocytogenes when data were expressed as multiples of the MIC.Numerical values of the pertinent pharmacological descriptors andstatistical analysis of their differences are shown in the Table.

Pertinent regression parameters^(a) (with confidence intervals [CI]),and statistical analysis of the dose-response curves illustrated in FIG.4.

L. monocytogenes EGD ⁺ antibiotic E^(max♦) (CI) EC₅₀ ^(⋄)(C1) C_(S)^(⋄⋄) R² CEM-101 −0.66 mg/L  0.020 0.11 0.934 (−1.28 to −0.037) (0.005to 0.073) a a x MIC 5.00 0.88 (1.36 to 18.5) a AZI −0.81 mg/L 2.66 11.60.953 (−2.11 to 0.48)  (0.91 to 7.73) a b x MIC 2.66 11.6 (0.81 to 3.1) a L. pneumophila ATCC 33153 ⁺⁺ antibiotic E_(max) ^(♦) (CI) EC₅₀ ^(⋄)(CI) C_(S) ^(⋄⋄) R² CEM-101 −1.03 mg/L   0.052 0.018 0.920 (−1.34 to−0.72) (0.012 to 0.23)  a a x MIC  13.1 4.56 (3.02 to 57.0) a AZI −0.83mg/L   2.86 2.90 0.903 (−2.00 to 0.34)  (0.17 to 48.6) a b x MIC 179.0181  (10.5 to 3038) b ^(a) using all data points shown in FIG. 4 (datafrom samples without antibiotics were not used because of evidence ofextracellular growth when the extracellular concentration of anantibiotic is lower than 0.01 x MIC (5). ⁺ original (post-phagocytosis)inoculum [time = Oh; CFU/mg protein]): L. monocytogenes, 1.67 ± 0.22 ×10⁶ (n = 3); L. pneumophila, 0.94 ± 0.60 × 10⁶. ^(♦)CFU decrease (inlogo units) at time = 24 h (L. monocytogenes) or 48 h (L. pneumophila)from the corresponding original inoculum, as extrapolated for antibioticconcentration ∞; samples yielding less than 5 counts were consideredbelow detection level, ^(⋄)concentration (in mg/L or in x MIC) causing areduction of the inoculum half-way between initial (E₀) and maximal(E_(max)) values, as obtained from the Hill equation (using a slopefactor of 1). ^(⋄⋄)concentration (in mg/L or in x MIC) resulting in noapparent bacterial growth (number of CFU identical to the originalinoculum), as determined by graphical intrapolation. Statisticalanalyses: analysis of the differences between the two antibiotics (percolumn for the corresponding rows; unpaired, two-tailed t-test): figureswith different lower case letters are significantly different from eachother (p < 0.05).

EXAMPLE. Dose-response studies in infected THP-1 macrophages Againstintraphagocytic S. aureus ATCC 25923, CEM-101 is more potent than AZI,CLR and TEL (lower Cs), In addition, CEM-101 is able to reduce theintracellular inoculum (E_(max)˜1 log), which is not observed with anyof AZI, CLR and TEL.

CEM-101 Uptake within Cells (ii): Role of the Cell Type

THP-1 J774 MDCK MDCK sur-expressing the Cells (human macrophages)(murine macrophages) (canine epith. cells) MDR1 efflux transportersCc/Ce ~50-150 ~60 ~45 ~30 at 5 h

EXAMPLE. Example Dose-response studies of CEM-101 vs. comparators (AZI,CLR and TEL) against intracellular S. aureus ATCC 25923 (THP-1macrophages). See FIG. 7 and the Table.

CEM-101 AZI CLR TEL Emax −0.80 + 0.11 0.04 ± 0.11 −0.18 ± 0.13 −0.29 ±0.16 Cs (mg/L) ~0.01 >50 ~0.86 ~0.27

EXAMPLE. Intracellular activity: comparative studies with otheranti-staphylococcal agents. Comparative dose-static response ofantibiotics against intracellular Staphylococcus aureus (strain ATCC25923) in THP-1 macrophages were measured. See FIG. 6 bars represent theMICs (in mg/L) or the extracellular static dose.

METHOD. Mouse peritoneal macrophages were infected with viable M.leprae, the drugs are added and incubated at 33° C. for 3 days. After 3days macrophages were lysed to release the intracellular M. leprae whichwere then assayed for viability by radiorespirometry and viabilitystaining. CEM-101 shows efficacy against intracellular M. lepraeviability.

The Thai-53 isolate of M. leprae, maintained by serial passages inathymic nu/nu mice footpads, was used for all experiments. For axenictesting freshly harvested viable M. leprae were incubated in mediumalong with different concentrations of the drugs (CEM-101, CLR andrifampin) for 7 days at 33° C. At the end of this incubationdrug-treated M. leprae were subjected to radiorespirometry to assessviability based on oxidation of palmitate and staining with viabilitydyes to assess the extent of membrane damage. For intracellular testingperitoneal macrophages from Swiss mice were infected with freshlyharvested viable M. leprae at an MOI of 20:1 for 12 hours. At the end ofthe infection extracellular bacteria were washed and drugs added atdifferent concentrations and incubated for 3 days at 33° C. At the endof 3 days cells were lysed to obtain the intracellular M. leprae forradiorespirometry and viability staining.

CEM-101 at 0.15 μg/ml was able to significantly (P<0.001) reduce theviability of M. leprae in both axenic and intracellular cultures whencompared to controls. Inhibition by CEM-101 was not statisticallydifferent from inhibition obtained with CLR under identical conditionsand at the same concentration.

EXAMPLE. The high potency of CEM-101 against Streptococcus pneumoniae,β-haemolytic and viridans group streptococci, Staphylococcus spp. andenterococci has been documented in early screening studies performedusing reference Clinical and Laboratory Standards Institute (CLSI)methods. Since mechanisms and occurrences of resistance are increasingrapidly that may compromise the MLSB-ketolide class, the bactericidalactivity (MBC and killing curves) of CEM-101 with five selected classesof antimicrobial agents when testing wild type (WT) andphenotypically/genotypically defined resistant organism subsets wasassessed. MBC determinations for CEM-101, TEL, and CLR used CLSI methodsfor 40 strains (6 species groups). KC used 8 strains (6 species groups).PAE was tested (5 strains) at 4× concentration for 1 or 2 hoursexposure; TEL control.

MBC and killing curve studies: A total of 40 strains (10 S. pneumoniae,10 S. aureus, and 5 each of β-haemolytic streptococci, viridans groupstreptococci, coagulase-negative staphylococci [CoNS] and enterococci)were MIC tested followed by MBC determinations using CLSI procedures(MIC and MBC range, 0.008-16 μg/ml). The lowest concentration of atested agent that killed ≧99.9% of the initial inoculum was defined asthe MBC endpoint (Tables 2 and 3). Time kill bactericidal activity wasperformed for CEM-101, TEL, CLR, and AZI on eight selected strainsaccording to methods described by Moody & Knapp, NCCLS M21-A3 and M26-A.The compounds were tested at 2×, 4×, 8×MIC; and colony counts wereperformed at T0, T2, T4, T8 and T24.

CEM-101 exhibited low MBC/MIC ratios (≦4) for BSA, SA andcoagulase-negative staphylococci; and 2-fold greater potency than TEL.SA, enterococci and some macrolide/CLN-resistant (R) strains had higherratios. KC results showed more rapid and greater cidal activity(concentration dependant) for CEM-101 compared to TEL. CEM-101 exhibitedcidal activity against several Gram-positive species at rates and anextent greater than TEL.

Distribution of Isolates According to MBC/MIC Ratio for CEM-101, TEL,CLR and AZI

No. of strains Organism/Antimicrobial agent with MBC/MIC value of: (no.tested) 1 2 4 8 16 ≧32 S. pneumoniae (10) CEM-101 3 5 0 0 0 2Telithromycin 2 6^(a) 0 0 0 2 Clarithromycin 2 3 1 0 0 —^(b)Azithromycin 2 4 0 0 0 —^(b) β-haemolytic streptococci (5) CEM-101 0 1 20 0 2 Telithromycin 0 1 1 1 0 2 Clarithromycin 0 0 1 1 0  2^(b)Azithromycin 0 0 0 0 2  2^(b) Viridans group streptococci (5) CEM-101 30 1 0 0 1 Telithromycin 2 1 1 0 0 1 Clarithromycin 0 0 1 0 0  3^(b)Azithromycin 0 0 0 0 1  3^(b) S. aureus (10) CEM-101 1 0 0 0 1 8Telithromycin 0 0 0 0 0 10  Clarithromycin 0 0 0 0 0  6^(b) Azithromycin0 0 0 0 0  6^(b) Coagulase-neg. staphylococci (5) CEM-101 1 1 0 3 0 0Telithromycin 0 0 0 0 2 3 Clarithromycin 0 0 0 0 0  4^(b) Azithromycin 00 0 0 0  4^(b) Enterococcus spp. (5) CEM-101 0 0 0 0 0 5 Telithromycin 00 0 0 0 5 Clarithromycin 0 0 0 0 0  2^(b) Azithromycin 0 0 0 0 0  2^(b)^(a)Includes six isolates with a MIC of ≦0.008 μg/ml and a MBC of 0.015μg/ml (off scale comparisons). ^(b)MBC was not evaluated on isolateswith resistant level MIC results.

CEM-101 showed rapid bactericidal activity (reduction of ≧3 log 10CFU/ml) against macrolide-susceptible strains of S. aureus, S.epidermidis, S. pneumoniae, S. pyogenes (only at 8×MIC) and viridansgroup streptococci, as well as a macrolide-resistant S. pyogenes.CEM-101 produced a greater reduction of CFU/ml and more rapid killingwhen compared to either TEL or the macrolides CLR and AZI.

Summary of Time Kill Curve Results

Organism Antimicrobial agent Antimicrobial activity S. aureus CEM-101Cidal at 2X, 4X, 8X (ATCC 29213) Telithromycin Cidal at 8X onlyClarithromycin Cidal at 8X only Azithromycin Cidal at 8X only S.epidermidis CEM-101 Cidal at 2X, 4X, 8X (095-2777A) Telithromycin StaticClarithromycin Static Azithromycin Static E. faecalis CEM-101 Static(ATCC 29212) Telithromycin Static Clarithromycin Static AzithromycinStatic S. pneumoniae CEM-101 Cidal at 2X, 4X, 8X (ATCC 49619)Telithromycin Cidal at 2X, 4X, 8X Clarithromycin Cidal at 2X, 4X, 8X(slow killing) Azithromycin Cidal at 2X, 4X, 8X (slow killing) S.pneumoniae CEM-101 Static (075-241B) Telithromycin Static S. pyogenesCEM-101 Cidal at 8X only (117-1612A) Telithromycin Cidal at 8X only(slow killing) Clarithromycin Cidal at 8X only (slow killing)Azithromycin Cidal at 8X only (slow killing) S. pyogenes CEM-101 Cidalat 2X, 4X, 8X (088-11708A) Telithromycin Cidal at 2X, 4X, 8X (slowkilling) S. mitis CEM-101 Cidal at 2X, 4X, 8X (112-1885A) TelithromycinCidal at 2X, 4X, 8X Clarithromycin Cidal at 8X only (slow killing)Azithromycin Cidal at 4X and 8X (slow killing)CEM-101 exhibited bactericidal activity when tested againstmacrolide-susceptible streptococci, CoNS and macrolide-resistantCLN-susceptible S. pneumoniae. CEM-101 MBC/MIC ratios can be high for S.aureus, but some strains showed MBC results remaining within thesusceptible range of concentrations.

EXAMPLE. Activity on Chlamydia. CEM-101, TEL, AZI, CLR, and doxycyclinewere provided as powders and solubilized according to the instructionsof the manufacturers. Drug suspensions were made fresh each time theassay was run.

C. pneumoniae: Isolates of C. pneumoniae tested included a referencestrain (TW 183), 9 isolates from children and adults with pneumonia fromthe United States (AR39, T2023, T2043, W6805, CWL 029, CM-1), an isolatefrom a child with pneumonia from Japan (J-21), and 2 strains frombronchoalveolar lavage specimens from patients with humanimmunodeficiency virus infection and pneumonia from the United States(BAL15 and BAL16).

C. trachomatis: 10 isolates of C. trachomatis, including standardisolates from the ATCC (E-BOUR, F-IC-CAL3, C-HAR32, J-UW-36, L2434,D-UW-57kx, B-HAR-36) and recent clinical isolates (N18 (cervical), N19(cervical), 7015 (infant eye))

In vitro susceptibility testing: Susceptibility testing of C. pneumoniaeand C. trachomatis was performed in cell culture using HEp-2 cells grownin 96-well microtiter plates. Each well was inoculated with 0.1 ml ofthe test strain diluted to yield 10³ to 10⁴ IFU/per ml, centrifuged at1,700×g for 1 hr. and incubated at 35° C. for 1 hr. Wells were aspiratedand overlaid with 0.2 mL of medium containing 1 μg of cycloheximide permL and serial two-fold dilutions of the test drug.

Duplicate plates were inoculated. After incubation at 35° C. for 48-72hrs, cultures were fixed and stained for inclusions withfluorescein-conjugated antibody to the lipopolysaccharide genus antigen(Pathfinder, Kallestad Diagnostics, Chaska, Minn.). The minimalinhibitory concentration (MIC) is the lowest antibiotic concentration atwhich no inclusions were seen. The minimal bactericidal concentration(MBC) was determined by aspirating the antibiotic containing medium,washing wells twice with phosphate buffered saline and addingantibiotic-free medium. Cultures were frozen at −70° C., thawed, passedonto new cells, incubated for 72 hrs then fixed and stained as above.The MBC is the lowest antibiotic concentration that results in noinclusions after passage. All tests were run in triplicate.

Activities of CEM-101 and Other Antibiotics Against 10 Isolates of C.pneumoniae

MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 101 0.25-1.00.25 0.25 0.25-1.0 0.25 Telithromycin 0.015-0.25 0.06 0.06 0.015-0.250.06 Azithromycin  0.015-0.125 0.125 0.125  0.015-0.125 0.125Clarithromycin  0.015-0.125 0.06 0.06  0.015-0.125 0.06 Doxycycline0.015-0.06 0.06 0.06 0.015-0.06 0.06

Activities of CEM-101 and Other Antibiotics Against 10 Isolates of C.trachomatis

MIC (μg/ml) MBC (μg/ml) Drug Range 50% 90% Range 90% CEM 101 0.125-0.5 0.25 0.25 0.125-0.5  0.25 Telithromycin 0.015-0.25  0.06 0.060.015-0.25  0.06 Azithromycin 0.015-0.125 0.125 0.125 0.015-0.125 0.125Clarithromycin 0.015-0.125 0.06 0.06 0.015-0.125 0.06 Doxycycline0.015-0.06  0.06 0.06 0.015-0.06  0.06The results of this study demonstrated that CEM-101 has in vitroactivity against C. trachomatis and C. pneumoniae comparable to othermacrolides and ketolides.

EXAMPLE. Tissue distribution. CEM-101 was well absorbed and distributedto the tissue. In the rat at 250 mg/kg/d, mean lung and liverconcentrations of CEM-101 were 17 and 15-fold higher than in plasma.Lung and liver concentrations were 503 and 711-fold higher than plasmaconcentrations at the 200 mg/kg/d dose in monkeys. Concentrations ofCEM-101 in the heart were significantly lower than levels found in lungor liver with levels 5 and 54-fold higher than plasma concentrations inrat and monkey, respectively.

What is claimed is:
 1. A method for treating a gastrointestinal diseasecaused by a bacteria in a host animal, the method comprising the step ofadministering an effective amount to the host animal of a compound ofthe formula

or a salt thereof, wherein: R₁₀ is hydrogen or acyl; W is H or F; A isCH₂; B is C₀-C₁₀ alkyl; and C is optionally substituted aryl oroptionally substituted heteroaryl.
 2. The method of claim 1 wherein R¹⁰is hydrogen.
 3. The method of claim 1 wherein W is F.
 4. The method ofclaim 1 wherein B is C₂-C₄ alkyl.
 5. The method of claim 1 wherein B isC₃ alkyl.
 6. The method of claim 1 wherein C is optionally substitutedaryl.
 7. The method of claim 1 wherein C is amino-substituted aryl. 8.The method of claim 1 wherein C is optionally substituted phenyl.
 9. Themethod of claim 1 wherein C is amino-substituted phenyl.
 10. The methodof claim 1 wherein C is 3-aminophenyl.
 11. The method of claim 1 whereinthe compound is CEM-101 or a salt thereof.
 12. The method of claim 1wherein the compound is CEM-101.
 13. The method of claim 11 wherein thegastrointestinal disease is chronic gastritis.
 14. The method of claim11 wherein the gastrointestinal disease is diarrhea.
 15. The method ofclaim 11 wherein the gastrointestinal disease is shigellosis.
 16. Themethod of claim 11 wherein the gastrointestinal disease is caused atleast in part by a bacteria selected from the group consisting ofSalmonella, Shigella, Staphylococcus, Campylobacter, Helicobacter,Clostridium, Enterococcus, Escherichia coli, Listeria, and Yersinia. 17.The method of claim 11 wherein the gastrointestinal disease is caused atleast in part by Enterococcus faecalis.
 18. The method of claim 11wherein the gastrointestinal disease is caused at least in part byYersinia enterocolitica.
 19. The method of claim 11 wherein thegastrointestinal disease is caused at least in part by Listeriamonocytogenes.
 20. The method of claim 11 wherein the gastrointestinaldisease is caused at least in part by Helicobacter pylori and thedisease is chronic gastritis.
 21. The method of claim 11 whereingastrointestinal disease is caused at least in part by Campylobacter andthe disease is diarrhea.
 22. The method of claim 11 wherein thegastrointestinal disease is caused at least in part by Salmonella andthe disease is diarrhea.
 23. The method of claim 11 wherein the bacteriais resistant to azithromycin.
 24. The method of claim 11 wherein thebacteria is resistant to clarithromycin.